DESCRIPTION: Biotinidase is the enzyme responsible for the recycling of the vitamin biotin from biocytin (epsilon-biotinyl-L-lysine) and biotinyl-peptides formed by the proteolytic degradation of biotin-dependent carboxylases. Biotinidase deficiency is the primary enzymatic defect in an inherited form of biotin-responsive, late-onset multiple carboxylase deficiency. The applicant has shown that in addition to its cleavage activity, biotinidase transfers biotin to various nucleophilic acceptors, including specific serum proteins, when incubated with biocytin at neutral to alkaline pH. His research group thus far has identified histones in this group of biotinylated protein in serum. In the proposed research, additional biotinylated proteins and small molecules will be identified in serum and in cells and tissues. It will also be determined if biotinidase biotinylates and/or removes biotin from biotin-dependent carboxylases and if it plays a role in transporting biotin into cells. To better understand these functions and the metabolism of biotinidase, the applicant plans to study the biochemistry and cell biology of the enzyme in normal serum and tissues, in various tumor cells, and in biotinidase-deficient states. The subcellular localization of biotinidase in normal and clinically relevant cultured cells and tissues will be determined using immunocytochemical techniques. The characteristics of uptake, turnover, and secretion of biotinidase by these cells will also be determined. Similar studies will also be performed using fibroblasts and lymphoblasts of individuals with biotinidase deficiency. The results will be compared to those of normal cells and will be correlated with the biochemical characteristics of biotinidase in sera of biotinidase-deficient individuals and with their molecular defects. These results will have implications for the prognosis and treatment of biotinidase-deficient children. In addition, the proposed research will provide a better understanding of the role of biotinidase in biotin metabolism and will be a paradigm for understanding the metabolism of other vitamins.